The CDC reports that at least 2 million infections and 23,000 deaths are caused annually by drug-resistant pathogens. One such pathogen, vancomycin-resistant enterococci (VRE), is a leading cause of hospital- associated infections and can become resistant to multiple antibiotics. Tigecycline (TGC), a tetracycline derivative, is a promising antibiotic for treating VRE infections. However, most VRE have the tetracycline- resistance gene tetM, which could pose a threat to the efficacy of TGC. Understanding the evolutionary dynamics leading to TGC resistance in an adapting population of vancomycin-resistant Enterococcus faecalis will be useful for developing strategies to maintain the effectiveness of this antibiotic of last resort. Our lab has developed techniques that use experimental evolution to model adaptation to drug resistance in the lab as it might occur in a patient. The goal of our work is to use these methods to identify the most important genomic and biochemical changes responsible for TGC resistance in E. faecalis. Over the course of 24 and 19 days, we performed two replicate adaption experiments where a population of pathogenic E. faecalis S613 was adapted from growth in 0.05 g/mL TGC to a final concentration of 1 g/mL TGC. Adaptation was facilitated using a novel bioreactor system that maintains a continuous culture at its fastest growth rate and exposes cells to increasing sub-minimal inhibitory concentrations of antibiotic. Whole genome sequencing of different strains isolated from the polymorphic end-point populations will be used to identify adaptive alleles and the linkages between different alleles. To survey the appearance and frequency of individual alleles within the population during adaptation, whole genome sequencing will be used on samples of the polymorphic population taken from each day of both adaptation experiments. Preliminary data suggest that adaptation is reproducible, as similar mutations reached high frequency by the end of both adaptation experiments. In preliminary studies, a 12-bp deletion in the ribosomal S10 gene reached fixation by the completion of the two replicate bioreactor adaptation experiments. We hypothesize that this S10 deletion could reduce the binding affinity of TGC for the ribosome, and we propose to do in vitro translation assays to determine the effect of this mutation on translation in the presence of TGC. Also, preliminary data from both experiments suggest that deletions up-regulating tetM expression and duplications of tetM were successful. Duplications of tetM occurred due to horizontal gene transfer of Tn916 (the conjugative transposon that carries tetM). Since Tn916 conjugation is coupled to tetM regulation, identifying the mechanisms of tetM regulation will be helpful in developing strategies not only to inhibit tetM expression, but also to deter Tn916-facilitated horizontal transfer of resistance. Therefore, we propose to study the mechanism of tetM regulation by probing the 5' untranslated region of tetM for a riboswitch using isothermal titration calorimetry. All together this work will provide important insights that can be used to identify the most successful adaptive trajectories leading to TGC resistance in E. faecalis.